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MCB > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

thinckness of band on southern blot means what?

A

amount of DNA present in band

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2
Q

what is RFLP used for?

A

to track a mutation in a family

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3
Q

allele markers (RFLP) are specific for what reason?

A

spouses come from different family background, if a spouse have the allele will not necessaryly have the mutation

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4
Q

ASO hybridization

A

based method to detect sequence varients (mutations of poly morphisms)

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5
Q

what type of prob is used in ASO?

A

oligonucleotides

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6
Q

how many probes to test for a gene?

A

2, one for wild tyoe one for mutatnt allele

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7
Q

what if theres not a hybridization in the mutatnt allele

A

negative result

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8
Q

type of target DNA used in ASO?

A

genomic DNA. dont need to run target DNA through a gel.

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9
Q

probes labeled with what?

A

p32

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10
Q

probes labeled with what?

A

p32

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11
Q

how many spots per individual in ASO?

A
  1. each with different probe
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12
Q

why short probes are used?

A

even with mismatch it will not bond. more specific results. if the probe is too long it will still bind even with mismatch

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13
Q

classic mutation used to talk about point mutation?

A

sickle cell disease

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14
Q

conservative point mutation

A

amino acid changed because of mutation will have similar properties with each other

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15
Q

non conservative mutation

A

amino acid replaced with another with different properties. ex. GLU to VAL

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16
Q

what types of questions on ASO hybridization?

A

only to analyse the dots. will not be tested on methods.

17
Q

how is dna amplified in pcr

A

exponentially.

18
Q

number of copies produced per dna molecule cycle?

A

2 to the power of number of cycles 2^n

19
Q

Taq polymerase

A

heat resistant polymerase from bacteria grwon in hot spring

20
Q

problem with Taq polymerase?

A

no proofreading

21
Q

advantage of Pfu polymerase?

A

has proofreading capacity, but it is slow

22
Q

PCR methods

A

start with dna polymerase, primers, dna sequence. and dNTP , magnesium

  1. heat to separate dna strands 95 degrees
  2. cool to 55-60 to allow primers to aneal
  3. dna polymerase from the 3 prime end of each primer at 72 degrees
23
Q

optimal temp. for Taq polymerase?

A

72 degrees

24
Q

themocyclers

A

metal block to insert test tubes equiped with timers and heating and cooling elements.
they automatically heat and cool solution.

25
Q

denaturation, anealing, and extension temperatures for pcr?

A

95, 55, 72, respectively

26
Q

what can pcr be used for?

A

RFLP
sequencing
direct mutation detection
determining repeat length

27
Q

limits for diagnostic of fragment size with pcr?

A

1 kb fragment

28
Q

how to desing pcr primers?

A
  1. have to have 2 primers, inside the region of interest. the primer is part of what is being synthesized
  2. place primer on 3’ end of each strand
    3
29
Q

general size of primer?

A

about 20 nt long

30
Q

how sequence of one strand relate to the other strand?

A

same as other. because other is complementary to it

31
Q

reverse primer sequence related to sequnce of other strand?

A

complementary.

32
Q

reverse primer sequence related to sequnce of other strand?

A

complementary.

33
Q

to use PCR to amplify RNA?

A

need to turn it into DNA first, resverse transcriptase to synthesize DNA from mRNA

34
Q

preparation of cDNA by PCR?

A

get mRNA and reverse transcriptase to rapidly get bunch of dna from it. no need to making cDNA library anymore

35
Q

RT-PCR?

A

reverse transcriptase PCR. product ismeasured as reaction is happening, not at the end.
measures the production of a fluorescent-labeled PCR produc in real time as the cycles are progressing

36
Q

Real time PCR or qPCR

A

quantitative method.

37
Q

is standard pcr quantitative? why

A

no, towards the end of reaction doubling is not perfect

MCB (6 decks)

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